Chemokine variants

ABSTRACT

The present invention provide the nucleotide and amino acid sequence of truncated RANTES (3-68) which has the same amino acid sequence as the wild-type RANTES, but with a serine/proline truncation at positions 1 and 2 from the N-terminus, respectively.

This is a 371 national phase application filed from and claims priorityto international patent application no. PCT/US98/25492, filed Dec. 1,1998, which claims priority from provisional patent application, U.S.Ser. No. 60/067,033 filed Dec. 1, 1997 which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to chemoattractant cytokines,called chemokines, and more specifically to truncated or variant formsof chemokines which have functions different from their wild-typecounterparts, methods of use and methods of producing such variantchemokines.

BACKGROUND OF THE INVENTION

Immunomodulatory proteins include chemotactic cytokines, called“chemokines”. Chemokines are small molecular weight immune ligands whichare chemoattractants for leukocytes, such as especially neutrophils,basophils, monocytes and T cells. There are two major classes ofchemokines which both contain four conserved cysteine residues whichform disulfide bonds in the tertiary structure of the proteins. The αclass is designated C-X-C (where X is any amilno acid), which includesIL-8, CTAP-III, gro/MGSA and ENA-78; and the β class, designated C-C,which includes MCP-1, MIP-1α and β, and regulated on activation, normalT expressed and secreted protein (RANTES). The designations of theclasses are according to whether an intervening residue spaces the firsttwo cysteines in the motif. In general, most C-X-C chemokines arechemoattractants for neutrophils but not monocytes, whereas C-Cchemokines appear to attract monocytes but not neutrophils. Recently, athird group of chemokines, the “C” group, was designated by thediscovery of a new protein called lymphotactin (Kelner, et al., Science,266:1395-1933, 1994). The chemokine family is believed to be criticallyimportant in the infiltration of lymphocytes and monocytes into sites ofinflammation.

Monocytes differentiate into macrophages as they migrate from the bloodto tissues during immune surveillance. At sites of inflammation,monocyte infiltration and macrophage accumulation are coordinated, inpart, by chemokines (1). The mechanisms that control the recruitment ofmonocytes and macrophages by chemoattractants have not been clearlydefined, but they may include regulation of the expression of chemokinesand their receptors (2) as well as the modification of chemokineactivity by posttranslational processing (3-5). Several chemokines sharea conserved NH2-X-Pro sequence (X, any amino acid) at the NH2-terminus(6), which conforms to the substrate specificity of dipeptidylexopeptidase IV (DPPIV) (7). DPPIV cleaves the first two amino acidsfrom peptides with penultimate proline or alanine residues, although nonatural substrate with immune function has been identified. This enzymeis also a leukocyte differentiation antigen, known as CD26 (8-10), thatis expressed on the cell surface mostly by T lymphocytes andmacrophages. Expression of CD26 has been associated with T cellactivation (8-10) and with susceptibility of a T cell line to infectionwith macrophage-tropic (M-tropic) HIV-1 (11).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that chemokines having aparticular N-terminal motif are natural substrates for a dipeptidyldipeptidase (DPPIV). Prior to the present invention, it was known thatCD26 is a leukocyte activation marker that possesses dipeptidylpeptidase IV (DPPIV) activity but natural substrates had not beenidentified. The present invention shows that several chemokines,including RANTES (regulated on activation, normal T expressed andsecreted) are substrates for recombinant soluble human CD26 (sCD26). Thepresent invention shows that DPPIV, e.g., CD26-mediated processing,together with cell activation induces changes in receptor expression andprovides a mechanism for differential cell recruitment and for theregulation of target cell specificity of chemokines.

Abbreviations: [Ca2+]i, cytosolic free Ca2+ concentration; DPPIV,dipeptidyl peptidase IV; ES-MS, electrospray mass spectromety; M-tropic,macrophage-tropic; pNA, p-nitroanilide; rh, recombinant human; sCD26,soluble CD26.

In a first embodiment, the invention provides the nucleotide and aminoacid sequence of truncated RANTES (3-68), which is the same as thewild-type RANTES with a Serine/Proline truncation at positions 1 and 2from the N-terminus, respectively.

In another embodiment, the invention provides a method for identifying acompound which modulates dipeptidyl peptidase IV (DPPIV)-mediatedchemokine processing. The method includes a) incubating componentscomprising the compound, DPPIV and a chemokine under conditionssufficient to allow the components to interact; and b) determining theN-terminal amino acid sequence of the chemokine before and afterincubating in the presence of the compound. Modulation of DPPIV-mediatedchemokine processing may be inhibition or stimulation of processing, forexample. Compounds which modulate such processing include peptides,peptidomimetics, and other small molecule compounds.

In another embodiment, the invention provides a method of inhibitingmembrane fusion between HIV and a target cell or between an HIV-infectedcell and a CD4 positive uninfected cell by contacting the target or CD4positive cell with a fusion-inhibiting effective amount of thepolypeptide of SEQ ID NO:2 (RANTES 3-68).

The invention also provides a method of treating a subject having or atrisk of having an HIV infection or disorder, including administering tothe subject, a therapeutically effective amount of a polypeptide of SEQID NO:2, wherein the polypeptide inhibits cell-cell fusion in cellsinfected with HIV The invention also provides a method of treating asubject having an HIV-related disorder associated with expression ofCCR5 comprising administering to an HIV infected or susceptible cell ofthe subject, a polypeptide of SEQ ID NO:2 or a nucleic acid sequenceencoding the polypeptide of SEQ ID NO:2 or other variant chemokine.Preferably, the subject is a human.

Also included are pharmaceutical compositions including the polypeptideof SEQ ID NO:2 or CD26, in pharmaceutically acceptable carriers.

In yet another embodiment, the invention provides a method for producinga variant chemokine having an activity different from the activity ofthe wild-type chemokine, including contacting the wild-type chemokinewith an N-terminal processing effective amount of dipeptidyl peptidaseIV (DPPIV), thereby truncating the chemokine and producing a variantchemokine. Chemokines may include, but are not limited to, RANTES,MIP-1, IP-10, eotaxin, MDC, and MCP-2.

The invention also provides a method for inhibiting HIV-1 replication ina host cell susceptible to HIV-1 infection, comprising contacting thecell or the host with an effective amount of dipeptidyl peptidase IV(DPPIV) enzyme such that macrophage-derived chemokine (MDC) is cleavedto produce truncated MDC, thereby providing antiviral activity andinhibiting HIV-1 replication and a A method for inhibiting HIV-1replication in a host cell susceptible to HIV-1 infection, comprisingcontacting the cell or the host with an effective amount of dipeptidylpeptidase IV (DPPIV) enzyme such that RANTES is cleaved to producetruncated RANTES, thereby providing antiviral activity and inhibitingHIV-1 replication.

In another embodiment, the invention provides a method for inhibitingdipeptidyl peptidase IV (DPPIV)-mediated chemokine processing comprisingcontacting DPPIV with an inhibiting effective amount of a compound whichinhibits DPPIV expression or activity.

In another embodiment, the invention provides a method for inhibiting anallergic or inflammatory reaction in a subject, comprising administeringto the subject an effective amount of Dipeptidyl peptidase IV (DPPIV)enzyme such that a chemokine is cleaved to produce a truncatedchemokine, thereby inhibiting an allergic or inflammatory reaction.Preferably, the chemokine is eotaxin.

In another embodiment, the invention provides a method for acceleratingangiogenesis or wound healing in a subject, comprising administering tothe subject an effective amount of an inhibitor of dipeptidyl peptidaseIV (DPPIV) enzyme activity or gene expression or a DPPIV-insensitivechemokine, such that chemolcine processing is inhibited, therebyaccelerating angiogenesis or wound healing. One exemplary chemokineuseful in the method for accelerating angiogenesis is IP-10.

In all of the above methods, the exemplary DPPIV shown in the presentinvention is CD26.

In yet another embodiment, the invention provides a method for diagnosisor prognosis of a subject having a chemokine-associated disorder. Themethod includes identifying the presence of a chemokine of interest froma specimen isolated from the subject; determining the amino-terminalsequence of the chemokine, wherein a full-length amino acid sequence isindicative of the presence of a wild-type chemokine polypeptide and atruncated amino-terminal sequence is indicative of the presence of avariant chemokine; and determining the concentration of wild-typechemokine as compared to variant chemokine, thereby providing adiagnosis of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. RANTES cleavage products after digestion with sCD26. RANTES wasincubated overnight with the indicated amounts of enzymatically active(E+) or enzymatically deficient (E( ) sCD26 and samples were subjectedto ES-MS analysis. The peaks in the spectrum at masses of 7905 to 7906and 7887 to 7890 are tentatively identified as [M+K+]+ of RANTES with(7904 daltons) and without (7886 daltons) a molecule of H2O,respectively; the labeled peaks at the left of the spectrum correspondto each of these molecular ions minus a Ser-Pro dipeptide (184 daltons).

FIG. 2. Competitive inhibition of DPPIV by RANTES(1-68). ColorimetricDPPIV enzyme assay was performed using human placental DPPIV and theGly-Pro-pNA substrate, in the presence or absence of the testcompetitors Ile-Pro-Ile, RANTES(1-68), or RANTES(3-68); the competitorconcentration is indicated on the horizontal axis. Data are means±SEM(n=3), except for the highest concentration of RANTES(1-68) andRANTES(3-68), for which only one sample was assayed in order to conservematerial. Similar results were obtained in a repeat experiment.

FIG. 3. RT-PCR analysis of chemokine receptor transcripts in monocytescultured in the absence (M) or presence (M+M-CSF) of M-CSF. Totalcellular RNA was subjected to RT-PCR analysis as described in Materialsand Methods. Control reactions performed without reverse transcriptasewere negative for each PCR product.

FIG. 4. Effects of chemokines on [Ca2+]i in monocytes cultured in theabsence (M) or presence (M+M-CSF) of M-CSF. Fura-2 labeled cells wereexposed (at the times indicated by arrowheads) to chemically synthesizedRANTES variants (100 nM) or other indicated rh chemokines (30 nM) (R & DSystems), and Ca2+ responses were measured. The final concentrations ofchemokines in this and subsequent experiments were sufficient to inducea maximal increase in [Ca2+]i in the responding cells, and furtherchallenge with the same dose produced little or no detectable change in[Ca2+]i. The duration (˜100 s) and amplitude (˜20 to 30% of Fura-2saturation) of Ca2+ responses were similar to those obtained forchemokines with human monocytes (36). Similar results were obtained intwo additional experiments.

FIG. 5. Desensitization of chemokine-induced Ca2+ responses byfull-length or truncated RANTES. Fura-2 labeled cells were stimulatedfirst with 100 nM RANTES(1-68) or RANTES(3-68), or were leftunstimulated. After ˜150 s, the cells were challenged with the RANTESvariants (100 nM) or other chemokines (30 nM) as indicated, and Ca2+responses were measured.

FIG. 6. Activity of flull-length and truncated RANTES in cellsexpressing recombinant CCR5 or CCR1 receptors. The [Ca2+]i was measuredin HEK-293 cells expressing CCR5 (A and C) and HOS-CD4 cells expressingCCR1 (B and D). (A and B) Cells were stimulated with variousconcentrations of the two RANTES variants as indicated and maximalfluorescence values were calculated from the peaks of the Ca2+ responsecurves. (C and D) Homologous and heterologous desensitization of theresponses induced by RANTES(1-68) and RANTES(3-68) was measured intransfectants as described in FIG. 5.

FIG. 7. Effects of full-length and truncated RANTES on HIV-1-inducedcytopathicity. (A) HOS-CD4.CCR5 cells were incubated with uninfected PM1cells or PM1 cells chronically infected with MV3-HXB2 virus in thepresence or absence of the indicated concentrations of RANTES variants.After 3 days, cell viability was measured by the XTT method. Data aremeans of triplicate samples (SEM, <20% of mean). (B) Representativephotomicrographs of HOS-CD4.CCR5 cells cultured with HIV-1-infected PM1cells in the absence or presence of RANTES (1-68) or RANTES(3-68) asindicated.

FIG. 8. The nucleotide and deduced amino acid sequences for RANTES 3-68(SEQ ID NO:1 and 2, respectively) are shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the discovery of variant forms ofchemokines which have different functions than their wild-typecounterparts. These variant chemokines are produced by cleavage with adipeptidyl peptidase IV (DPPIV) which cleaves at the N-terminus of apolypeptide when there is a proline or an alanine at position 2.

Overview

CD26 is a leukocyte activation marker that possesses dipeptidylpeptidase IV (DPPIV) activity but whose natural substrates andimmunological functions have not been clearly defined. Severalchemokines, including RANTES (regulated on activation, normal Texpressed and secreted) have now been shown to be substrates forrecombinant soluble human CD26 (sCD26). The truncated RANTES(3-68)lacked the ability of native RANTES(1-68) to increase the cytosoliccalcium concentration in human monocytes, but it still induced thisresponse in macrophages activated with macrophage colony-stimulatingfactor (M-CSF). Analysis of chemokine receptor messenger RNAs andpatterns of desensitization of chemokine responses showed that thedifferential activity of the truncated molecule results from an alteredreceptor specificity. RANTES(3-68) showed a reduced activity, relativeto that of RANTES(1-68), with cells expressing the recombinant CCR1chemokine receptor, but it retained the ability to stimulate CCR5receptors and to inhibit the cytopathic effects of HIV-1. Our resultsindicate that CD26-mediated processing together with cell activationinduced changes in receptor expression provide an integrated mechanismfor differential cell recruitment and for the regulation of target cellspecificity of RANTES, and possibly other chemokines.

Nucleotide and Amino Acid Sequences of RANTES Variant (3-68) or OtherChemokine Variants

In a first embodiment, the invention provides a substantially purifiedRANTES variant polypeptide exemplified by the amino acid sequence of SEQID NO:2. The term “polypeptide” means any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation), and includes natural proteins as wellas synthetic or recombinant polypeptides and peptides.

The term “substantially pure” as used herein refers to RANTES (3-68) orother variant chemokine which is substantially free of other proteins,lipids, carbohydrates or other materials with which it is naturallyassociated. One skilled in the art can purify RANTES (3-68) or othervariant chemokine using standard techniques for protein purification.The substantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the RANTES (3-68) orother variant chemokine polypeptide can also be determined byamino-terminal amino acid sequence analysis. RANTES (3-68) or othervariant chemoline polypeptide includes functional fragments of thepolypeptide, as long as the activity of RANTES (3-68) or other variantchemokine remains. Such functional variants would include the N-terminuswhich is truncated as compared to the wild-type RANTES or otherchemokine. The term “variant” as used herein refers to a polypeptidehaving substantially the same polypeptide sequence as the correspondingwild-type polypeptide, with minor amino acid variations. These aminoacid variations result in a polypeptide having various additional and/ordifferent functions from the wild-type polypeptide, and possibly havingaltered receptor specificity as compared to the wild-type polypeptide.Smaller peptides containing the biological activity of RANTES (3-68) orother variant chemokine are included in the invention. The term“substantially pure,” when referring to an chemokine polypeptide, meansa polypeptide that is at least 60%, by weight, free from the proteinsand naturally-occurring organic molecules with which it is naturallyassociated. A substantially pure RANTES (3-68) or other variantchemokine polypeptide is at least 75%, more preferably at least 90%, andmost preferably at least 99%, by weight, RANTES (3-68) or other variantchemokine polypeptide. A substantially pure RANTES (3-68) or othervariant chemokine can be obtained, for example, by extraction from anatural source; by expression of a recombinant nucleic acid encoding aRANTES (3-68) or other variant chemokine polypeptide, or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, e.g., column chromatography, polyacrylamide gel electrophoresis,or HPLC analysis.

Minor modifications of the recombinant RANTES (3-68) or other variantchemokine primary amino acid sequence may result in proteins which havesubstantially equivalent activity as compared to the RANTES (3-68) orother variant chemokine polypeptide described herein. Such modificationsmay be deliberate, as by site-directed mutagenesis, or may bespontaneous. All of the polypeptides produced by these modifications areincluded herein as long as the biological activity of RANTES (3-68) orother variant chemokine still exists. Further, deletion of one or moreamino acids can also result in a modification of the structure of theresultant molecule without significantly altering its biologicalactivity. This can lead to the development of a smaller active moleculewhich would have broader utility.

The polynucleotide sequence encoding the RANTES (3-68) or other variantchemokine polypeptide of the invention includes the disclosed sequenceand conservative variations thereof. The term “conservative variation”as used herein denotes the replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine, and the like. The term “conservative variation” alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

The invention provides isolated polynucleotides encoding the RANTES(3-68) or other variant chemokine polypeptide. In one embodiment, thepolynucleotide is the nucleotide sequence of SEQ ID NO:1. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode RANTES(3-68) or other variant chemokine. It is understood that allpolynucleotides encoding all or a portion of RANTES (3-68) or othervariant chemokine are also included herein, as long as they encode apolypeptide with RANTES (3-68) or other variant chemokine activity(e.g., does not bind to CCR1 but binds to CCR5). Such polynucleotidesinclude naturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, RANTES (3-68) or other variant chemokinepolynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for RANTES (3-68) or other variant chemokinealso includes antisense sequences. The polynucleotides of the inventioninclude sequences that are degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of RANTES(3-68) or other variant chemokine polypeptide encoded by the nucleotidesequence is functionally unchanged. Abbreviations for the amino acidresidues are follows: A, Ala; C, Cys; D, Asp: E, Glu: F, Phe; G, Gly; H,His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

As used herein, “polynucleotide” also refers to a nucleic acid sequenceof deoxyribonucleotides or ribonucleotides in the form of a separatefragment or a component of a larger construct. DNA encoding portions orall of the polypeptides of the invention can be assembled from cDNAfragments or from oligonucleotides that provide a synthetic gene whichcan be expressed in a recombinant transcriptional unit.

An isolated polynucleotide as described herein is a nucleic acidmolecule that is separated in some way from sequences in the naturallyoccurring genome of an organism. Thus, the term “isolatedpolynucleotide” includes any nucleic acid molecules that are notnaturally occurring. The term therefore includes, for example, arecombinant polynucleotide which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate moleculeindependent of other sequences. It also includes a recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequences.

Specifically disclosed herein is a DNA sequence containing the RANTESpolypeptide gene encoding RANTES truncated at positions 1 and 2. Thepolynucleotide encoding RANTES (3-68) includes FIG. 8 (SEQ ID NO:1), aswell as nucleic acid sequences complementary to SEQ ID NO:1. Acomplementary sequence may include an antisense nucleotide. When thesequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID NO:1 arereplaced by ribonucleotides A, G, C, and U, respectively. Also includedin the invention are fragments of the above-described nucleic acidsequences that are at least 15 bases in length, which is sufficient topermit the fragment to selectively hybridize to DNA that encodes theprotein of SEQ ID NO: 2 under physiological conditions or a close familymember of RANTES. The term “selectively hybridize” refers tohybridization under moderately or highly stringent conditions whichexcludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the RANTES (3-68) or other variant chemokine polynucleotideof the invention is derived from a mammalian organism, and mostpreferably from a mouse, rat, or human. Screening procedures which relyon nucleic acid hybridization make it possible to isolate any genesequence from any organism, provided the appropriate probe is available.Oligonucleotide probes, which correspond to a part of the sequenceencoding the protein in question, can be synthesized chemically. Thisrequires that short, oligopeptide stretches of amino acid sequence mustbe known. The DNA sequence encoding the protein can be deduced from thegenetic code, however, the degeneracy of the code must be taken intoaccount. It is possible to perform a mixed addition reaction when thesequence is degenerate. This includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful in thedetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981; Maniatis, et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y 1989).

The development of specific DNA sequences encoding RANTES (3-68) orother variant chemokine can also be obtained by: 1) isolation ofdouble-stranded DNA sequences from the genomic DNA; 2) chemicalmanufacture of a DNA sequence to provide the necessary codons for thepolypeptide of interest; and 3) in vitro synthesis of a double-strandedDNA sequence by reverse transcription of mRNA isolated from a eukaryoticdonor cell. In the latter case, a double-stranded DNA complement of mRNAis eventually formed which is generally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gtl1, can be screenedindirectly for RANTES (3-68) or other variant chemokine peptides havingat least one epitope, using antibodies specific for RANTES (3-68) orother variant chemokine. Such antibodies can be either polyclonally ormonoclonally derived and used to detect expression product indicative ofthe presence of RANTES (3-68) or other variant chemokine cDNA.

The isolated polynucleotide sequences of the invention also includesequences complementary to the polynucleotides encoding RANTES (3-68) orother variant chemokine (antisense sequences). Antisense nucleic acidsare DNA or RNA molecules that are complementary to at least a portion ofa specific mRNA molecule (Weintraub et al., Scientific American 262:40,1990). The invention includes all antisense polynucleotides that inhibitproduction of RANTES (3-68) or other variant chemokine polypeptides. Inthe cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized andintroduced into a target RANTES (3-68) or other variantchemokine-producing cell. The use of antisense methods to inhibit thetranslation of genes is known in the art, and is described, e.g., inMarcus-Sakura (Anal. Biochem., 172:289, 1988).

In addition, ribozyme nucleotide sequences for RANTES (3-68) or othervariant chemokine are included in the invention. Ribozymes are RNAmolecules possessing the ability to specifically cleave othersingle-stranded RNA in a manner analogous to DNA restrictionendonucleases. Through the modification of nucleotide sequences encodingthese RNAs, molecules can be engineered to recognize specific nucleotidesequences in an RNA molecule and cleave it (Cech (1988) J. Amer. Med.Assn. 260:3030). A major advantage of this approach is that, becausethey are sequence-specific, only mRNAs with particular sequences areinactivated.

There are two basic types of ribozymes, tetrahymena-type (Hasselhoff(1988) Nature 334:585) and “hammerhead”-type. Tetrahymena-type ribozymesrecognize sequences which are four bases in length, while“hammerhead”-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences.

DNA sequences encoding RANTES (3-68) or other variant chemokine can beexpressed in vitro by DNA transfer into a suitable host cell. “Hostcells” are cells in which a vector can be propagated and its DNAexpressed. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication.However, such progeny are included when the term “host cell” is used.Methods of stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

In the present invention, the RANTES (3-68) or other variant chemokinepolynucleotide sequences may be inserted into a recombinant expressionvector. The term “recombinant expression vector” refers to a plasmid,virus or other vehicle known in the art that has been manipulated byinsertion or incorporation of the RANTES (3-68) or other variantchemokine genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, m etallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding RANTES (3-68) or other variantchemokine can be expressed in either prokaryote or eukaryotes. Hosts caninclude microbial, yeast, insect and mammalian organisms. Methods ofexpressing DNA sequences having eukaryotic or viral sequences inprokaryote are well known in the art. Biologically functional viral andplasmid DNA vectors capable of expression and replication in a host areknown in the art. Such vectors are used to incorporate DNA sequences ofthe invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the RANTES (3-68) or othervariant chemokine of the invention, and a second foreign DNA moleculeencoding a selectable phenotype, such as the herpes simplex thymidinekinase gene. Another method is to use a eukaryotic viral vector, such assimian virus 40 (SV40) or bovine papilloma virus, to transiently infector transform eukaryotic cells and express the protein. (see for example,Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed.,1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

Antibodies That Distinguish Wild-type Chemokine from Truncated Chemokine

The present invention also provides antibodies useful for distinguishingbetween wild-type and DPPIV-truncated chemokine polypeptides.Preferably, the antibodies are produced by using N-terminal peptideshaving about 8 or more amino acids. Therefore, antibodies produced willdistinguish between a chemokine, such as RANTES, that containsN-terminal amino acids, and a chemokine that has been cleaved, forexample by CD26. The preparation of polyclonal antibodies is well-knownto those skilled in the art. See, for example, Green et al., Productionof Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages1-5 (Humana Press 1992); Coligan et al., Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS INIMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated byreference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORYMANUAL, page 726 (Cold Spring Harbor Pub. 1988), which are herebyincorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See,e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., Purification of Immunoglobulin G (IgG), in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992). Methods ofin vitro and in vivo multiplication of monoclonal antibodies iswell-known to those skilled in the art. Multiplication in vitro may becarried out in suitable culture media such as Dulbecco's Modified EagleMedium or RPMI 1640 medium, optionally replenished by a mammalian serumsuch as fetal calf serum or trace elements and growth-sustainingsupplements such as normal mouse peritoneal exudate cells, spleen cells,bone marrow macrophages. Production in vitro provides relatively pureantibody preparations and allows scale-up to yield large amounts of thedesired antibodies. Large scale hybridoma cultivation can be carried outby homogenous suspension culture in an airlift reactor, in a continuousstirrer reactor, or in immobilized or entrapped cell culture.Multiplication in vivo may be carried out by injecting cell clones intomammals histocompatible with the parent cells, e.g., osyngeneic mice, tocause growth of antibody-producing tumors. Optionally, the animals areprimed with a hydrocarbon, especially oils such as pristane(tetramethylpentadecane) prior to injection. After one to three weeks,the desired monoclonal antibody is recovered from the body fluid of theanimal.

Therapeutic applications for antibodies disclosed herein are also partof the present invention. For example, antibodies of the presentinvention may also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in Goldenberg et al., International PatentPublication WO 91/11465 (1991) and Losman et al., Int. J. Cancer 46:310(1990), which are hereby incorporated by reference.

Alternatively, a therapeutically useful anti-RANTES (3-68) or othervariant chemokine antibody may be derived from a “humanized” monoclonalantibody. Humanized monoclonal antibodies are produced by transferringmouse complementarity determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, by Jones et al., Nature 321: 522 (1986); Riechmann et al.,Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988);Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit.Rev. Biotech. 12: 437 (1992); and Singer et al., J. Immunol. 150: 2844(1993), which are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433(1994), which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856(1994); and Taylor et al., Int. Immunol. 6:579 (1994), which are herebyincorporated by reference.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference. See also Nisonhoff etal., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119(1959); Edelman et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422(Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent, as described in Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,supra. Preferably, the Fv fragments comprise V_(H) and V_(L) chainsconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are described, for example, by Whitlow et al., METHODS: A COMPANIONTO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al., Science242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et al.,Bio/Technology 11: 1271-77 (1993); and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Lanick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

Screen for Compounds Which Modulate DDPPIV

In another embodiment, the invention provides a method for identifying acompound which modulates dipeptidyl peptidase IV (DPPIV)-mediatedchemokine processing. The method includes: a) incubating componentscomprising the compound, DPPIV and a chemokine under conditionssufficient to allow the components to interact; and b) determining theN-terminal amino acid sequence of the chemokine before and afterincubating in the presence of the compound. Compounds that inhibit DPPIVinclude peptides, peptidomimetics, polypeptides, chemical compounds andbiologic agents. Preferably the DPPIV is CD26. If a compound inhibitsthe DPPIV or CD26 enzymatic activity, the chemokine will have anN-terminal amino acid sequence which corresponds to the wild-typepolypeptide. Alternatively, if the compound stimulates DPPIV or CD26enzymatic activity, the chemokine will have a truncated amino-terminalamino acid sequence. The amino acid sequence can be determined bystandard N-terminal sequencing methods or by contacting the chemokinewith a monoclonal antibody which distinguishes between wild-type andtruncated or variant chemokine, for example.

Incubating includes conditions which allow contact between the testcompound and the chemokine and a DPPIV. Contacting includes in solutionand in solid phase, or in a cell. The test compound may optionally be acombinatorial library for screening a plurality of compounds. Compoundsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs)(Landegren, et al., Science, 241:1077, 1988), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren, et al.,Science, 242:229-237, 1988).

Methods for Producing Variant Chemokines

In another embodiment, the invention provides a method for producing avariant chemokine having an activity different from the activity of thewild-type chemokine, including contacting the wild-type chemokine withan N-terminal processing effective amount of dipeptidyl peptidase IV(DPPIV), thereby truncating the chemokine and producing a variantchemokine. The term “N-terminal processing effective amount” refers tothat amount of a DPPIV that cleaves the amino terminus of a wild-typechemokine polypeptide to produce a chemokine lacking the first two aminoterminal amino acids. For example, incubation of RANTES with an“N-terminal processing effective amount” of CD26 results in RANTES(3-68) which has different activity than wild type RANTES. Chemokinesthat contain amino acid motifs at the N-terminus include but are notlimited to RANTES, MIP-1, IP-10, eotaxin, macrophage-derived chemokine(MDC) and MCP-2. Other chemokines known in the art can be assessed forsensitivity to cleavage by DPPIVs as described herein by determining thefirst two amino terminal amino acids.

Contacting the chemokine can be in vitro or in vivo. For example, aspecimen isolated from a subject, such as a human, or a mixture or puresample of chemokine, can be contacted with DPPIV in vitro. Thecontacting of the DPPIV and chemokine is deemed sufficient when cleavageof the chemokine has occurred. It may be desirable to only cleave afraction of the total chemokine population, therefore, samples can beanalyzed at various time of incubation to determine the optimalconditions for the desired concentration of wild-type versus truncatedvariant chemokine achieved.

The preferred chemokine illustrated herein is RANTES and the preferredDPPIV is CD26. Other chemokines and DPPIVs are also included in themethod of the invention.

Inhibition of DPPIV

In another embodiment, the invention provides a method for inhibitingdipeptidyl peptidase IV (DPPIV)-mediated chemokine processing comprisingcontacting DPPIV with an inhibiting effective amount of a compound whichinhibits DPPIV expression or activity. For example, the method includesinhibiting CD26 expression or activity. To determine whether the DPPIVactivity or expression is inhibited, an assay to detect cleavage of achemokine having an alanine or proline at position 2, or a Northern blotanalysis, can be performed, respectively. Other standard methods can beused to detect inhition of gene expression or enzymatic activity. Forexample, incubation of CD26, RANTES and a compound suspected ofinhibiting CD26 activity, would result in wild-type RANTES, but littleor no cleaved RANTES (or RANTES “variant”).

Methods of Use for Inhibiting HIV-1 Replication, Allergic orInflammatory Reactions, and Angiogenesis

In another embodiment, the invention provides a method for inhibitingHIV-1 replication in a host cell susceptible to HIV-1 infection,comprising contacting the cell or the host with an effective amount ofdipeptidyl peptidase IV (DPPIV) enzyme such that macrophage-derivedchemokine (MDC) or RANTES is cleaved to produce truncated MDC or RANTES,respectively, thereby providing antiviral activity and inhibiting HIV-1replication. The present invention provides data demonstrating thatcleaved RANTES blocks HIV-1 infection (EXAMPLE 7). While not wanting tobe bound to a particular theory, it is believed that the activity of MDCis increased upon cleavage. MDC suppresses HIV-1 replication, thus, itis desirable for AIDS patients, or individuals at risk of HIV-1infection to have increased levels of cleaved MDC. Other chemokines mayalso be useful in the method of the invention fro inhibiting HIV-1replication.

In yet another embodiment, the invention provides a method forinhibiting an allergic or inflammatory reaction in a subject, comprisingadministering to the subject an effective amount of dipeptidyl peptidaseIV (DPPIV) enzyme such that a chemokine is cleaved to produce atruncated chemokine, thereby inhibiting an allergic or inflammatoryreaction. Preferably, a chemokine useful for inhibition allergic orinflammatory reactions is a truncated eotaxin.

The use of a truncated chemokine in the method of the invention mayinhibit or depress an immune or inflammatory response where desirable,such as in graft rejection responses after organ and tissuetransplantations, or autoimmune disease. Some of the commonly performedtransplantation surgery today includes organs and tissues such askidneys, hearts, livers, skin, pancreatic islets and bone marrow.However, in situations where the donors and recipients are notgenetically identical, graft rejections can still occur. Autoimmunedisorders refer to a group of diseases that are caused by reactions ofthe immune system to self antigens leading to tissue destruction. Theseresponses may be mediated by antibodies, auto-reactive T cells or both.Some important autoimmune diseases include diabetes, autoimmunethyroiditis, multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosis, and myasthenia gravis. Other allergic or inflammatoryresponses are included in the method of the invention.

In another embodiment, the invention provides a method for acceleratingangiogenesis or wound healing in a subject, comprising administering tothe subject an effective amount of an inhibitor of dipeptidyl peptidaseIV (DPPIV) enzyme activity or gene expression or a DPPIV-insensitivechemokine, such that chemokine processing is inhibited, therebyaccelerating angiogenesis or wound healing. For example, new bloodvessels are required for tissue repair and enhanced blood vessel growthmay aid in improving circulation to ischemic limbs and heart tissuesuffering from atherosclerotic disease, healing skin ulcers or otherwounds, and establishing tissue grafts. Preferably, a chemokine usefulfor accelerating angiogenesis is a wild-type IP-10. Cleavage of IP-10appears to inactivate the activity of IP-10, therefore it is desirableto inhibit cleavage of IP-10. Alternatively, it may be desirable toprovide a variant IP-10 polypeptide which contains an amino acidsubstitution at position 2, such that neither proline nor alanine ispresent, which would result in a DPPIV-insensitive chemokine. However,such a variant must retain the activity of wild-type IP-10, e.g., achemoattractant for NK cells.

Methods of Diagnosis of Chemokine-associated Disorders

In another embodiment, the invention provides a method for diagnosis andprognosis of chemokine-associated disorders. The method includesidentifying the presence of a chemokine of interest from a specimenisolated from the subject; determining the amino-terminal sequence ofthe chemokine, wherein a full-length amino acid sequence is indicativeof the presence of a wild-type chemokine polypeptide and a truncatedamino-terminal sequence is indicative of the presence of a variantchemokine; and determining the concentration of wild-type chemokine ascompared to variant chemokine, thereby providing a diagnosis of thesubject. This method is also useful for prognosis of a subject, forexample, a subject having AIDS and being treated with a particulartherapeutic regimen. The amino-terminal sequence of the chemokine isdetermined, for example, by standard N-terminal sequencing, or bycontacting the chemokine with an antibody which distinguishes wild-typefrom variant chemokine polypeptide, as described above. Use ofmonoclonal antibodies, for example, allows simple detection by ELISA orother methods. Specimens useful for such diagnosis include but are notlimited to blood, sputum, urine, saliva, cerebrospinal fluid, and serum.

Pharmaceutical Compositions

The invention also includes various pharmaceutical compositions that areuseful for therapeutic applications as described herein. Thepharmaceutical compositions according to the invention are prepared bybringing a polypeptide such as SEQ ID NO:2 (RANTES (3-68)) or a DPPIV,such as CD26, into a form suitable for administration to a subject usingcarriers, excipients and additives or auxiliaries. Frequently usedcarriers or auxiliaries include magnesium carbonate, titanium dioxide,lactose, mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

In another embodiment, the invention relates to a method of treating asubject having an HIV-related disorder associated with expression ofCCR5 including administering to an HIV-infected or susceptible cell of asubject a therapeutically effective dose of a pharmaceutical compositioncontaining the compounds of the present invention and a pharmaceuticallyacceptable carrier. “Administering” the pharmaceutical composition ofthe present invention may be accomplished by any means known to theskilled artisan. By “subject” is meant any mammal, preferably a human.Such a method can be performed in vivo or ex vivo for example. Forexample, a vector containing a nucleic acid sequence encoding SEQ IDNO:2 or another truncated chemokine can be utilized for introducing thecomposition into a cell of the subject.

In another embodiment, the invention provides a method of treating asubject having or at risk of having an HIV infection or disorder,comprising administering to the subject, a therapeutically effectiveamount of a polypeptide of SEQ ID NO:2, wherein the polypeptide inhibitscell-cell fusion in cells infected with HIV. This method is performed asdiscussed above.

In another embodiment, the invention provides a method of inhibitingmembrane fusion between HIV and a target cell or between an HIV-infectedcell and a CD4 positive uninfected cell comprising contacting the targetor CD4 positive cell with a fusion-inhibiting effective amount of thepolypeptide of SEQ ID NO:2.

The pharmaceutical compositions are preferably prepared and administeredin dose units. Solid dose units are tablets, capsules and suppositories.For treatment of a patient, depending on activity of the compound,manner of administration, nature and severity of the disorder, age andbody weight of the patient, different daily doses are necessary. Undercertain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention are ingeneral administered topically, intravenously, orally or parenterally oras implants, but even rectal use is possible in principle. Suitablesolid or liquid pharmaceutical preparation forms are, for example,granules powders, tablets, coated tablets, (micro)capsules,suppositories, syrups, emulsions, suspensions, creams, aerosols, dropsor injectable solution in ampule form and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners orsolubilizers are customarily used as described above. The pharmaceuticalcompositions are suitable for use in a variety of drug delivery systems.For a brief review of present methods for drug delivery, see Langer,Science, 249: 1527-1533 (1990), which is incorporated herein byreference.

The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Gilman et al. (eds.)(1990) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS,8th ed., Pergamon Press; and REMINGTON'S PHARMACEUTICAL SCIENCES, 17thed. (1 990), Mack Publishing Co., Easton, Pa., each of which is hereinincorporated by reference.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

EXAMPLE 1 Materials and Methods

Cell cultures and transfections. Monocytes were isolated from humanPBMCs of healthy donors by counter-current centrifugal elutriationMonocyte-derived macrophages were prepared by culturing monocytes for 6days at a density of 106 cells/ml in serum-free macrophage medium (GibcoBRL, Grand Island, N.Y.) supplemented with recombinant human (rh) M-CSF(10 ng/ml) (R&D Systems, Minneapolis, Minn.).

Human embryonic kidney (HEK)-293 cells grown to confluence in DMEMsupplemented with 10% heat-inactivated FCS, penicillin, streptomycin, 2mM glutamine, and 10 mM Hepes (pH 7.4) were transfected with plasmid DNAencoding CCR5 (12). CD4-positive human osteosarcoma (HOS-CD4) cell linestransfected with individual chemokine receptor cDNAs were obtained fromN. Landau, and were grown in the above culture medium supplemented withpuromycin.

The derivative of the PM1 cell line chronically infected with therecombinant HIV-1 clone MV3-HXB2 has been described previously (11).sCD26 cleavage and electrospray mass spectrometry (ES-MS). To create therecombinant soluble human CD26 (sCD26) construct, a signal peptidasecleavage consensus sequence was introduced in the pTZ-CD26.11 cDNA (13)by a Leu to Ala substitution at residue 28. To obtain enzyme negativeconstruct, the Ser at residue 630 was further replaced by Ala. The twoconstructs were cloned into the pEE14.HCMV expression vector andtransfected into CHO-K1 cells (14). The enzymatically active (E+) andenzymatically deficient (E−) sCD26 proteins were purified from cellculture supernatants of stable transfectants, and were tested in Westernblotting and DPPIV enzyme assays (15). Both proteins had a relativemolecular weight of 110 kDa, bound equally well to several CD26 mAbs,but only the E+ sCD26 showed detectable DPPIV activity. rhRANTES, MCP-1,MCP-2, eotaxin, and IP-10 (100 nM) (Peprotech, Rocky Hill, N.J.) wereincubated overnight at 37° C. with different amounts of E+ or E(sCD26 in50 (l of PBS. Samples were desalted and concentrated by using a peptideWap (Michrom BioResources, Inc., Auburn, Calif.), or a reversed-phase(RP) HPLC interface. ES-MS analysis of samples was performed in 50%acetonitrile, supplemented with 0.1% (v/v) glacial acetic acid, using aFinnigan (San Jose, Calif.) TSQ 7000 triple-stage quadrupole massspectrometer. Several scans were summed to obtain the final spectrum.Peptide synthesis. Full-length and truncated RANTES were synthesizedwith an Applied Biosystems (Foster City, Calif.) peptide synthesizeraccording to fluorenyl methoxycarbonyl (FMOC) chemistry. FMOC-protectedamino acids were added stepwise with ninhydrin monitoring at each cycle.The peptides were folded by air oxidation and purified by RP HPLC.Peptide sequences were confirmed by amino acid analysis and Edmansequence analysis, and the molecular masses were confirmed by ES-MSanalysis. There was no substantial difference in the activities ofchemically synthesized full-length RANTES and rhRANTES(1-68) as judgedby the Ca2+ influx and anti-HIV-1 assays used in this study.

Colorimetric DPPIV Enzyme Assay

The p-nitroanilide (pNA)-conjugated Gly-Pro dipeptide substrate and testcompetitors were mixed and added to human placental DPPIV (Enzyme SystemProducts), and the resulting mixture was incubated at room temperaturein a final volume of 150 (1 containing 50 mM tris-HCl (pH 8.0) and 0.15M NaCl. The final concentrations of DPPIV and Gly-Pro-pNA were 1.25mU/ml and 400 (M, respectively. The kinetics of the enzyme reaction weremonitored by measuring absorbance at 405 nm with a Vmax kineticmicroplate reader (Molecular Devices, Menlo Park Calif.). The percentageinhibition of enzyme activity was calculated from the maximal velocityfor each sample and from that apparent in the absence of competitor(100% activity).

RT-PCR analysis. Isolated total cellular RNA of monocytes was subjectedto first-strand cDNA synthesis. PCR amplification of cDNA was performedfor 30 cycles (92° C. for 1 min, 40° C. for 1 min, 72° C. for 1 min)with primers specific for CCR1, CCR2b, CCR3, CCR5, CXCR4, and GAPDH.Separated products were stained with SYBR Green I (Molecular Probes,Eugene, Oreg.).

Cytosolic Calcium Measurements

Cells (107/ml) were washed and incubated in the dark at 37° C. for 45min in Ca2+ buffer [136 mM NaCl, 4.8 mM KCl, 5 mM glucose, 1 mM CaCl2,20 mM Hepes (pH 7.4)] supplemented with 5 (M Fura-2 acetoxymethyl esterthat had been premixed with 10% Pluronic<< F-127 (Molecular Probes). Thecells were then washed and resuspended at 2 (106 cells/ml in Ca2+ buffercontaining BSA (1 mg/ml), and portions (2 ml) of the cell suspensionwere exposed at different time points in a stirred cuvette at 37° C. tochemokines. Fluorescence was monitored with a Photon TechnologyInternational d scan (South Brunswick, N.J.), and data were recorded asthe relative ratio of fluorescence at excitation wavelengths of 340 and380 nm, with emission measured at 510 nm. After each measurement,maximal and minimal fluorescence were assessed by addition of 20 (Mionomycin followed by 5 mM MnCl2. Assay for HIV-1-induced cytopathicity.HOS-CD4.CCR5 cells (2 (104) were incubated for 1 hour at 37° C. withRANTES variants in 150 (1 of culture medium containing 20% FCS, and werethen mixed with 50 (1 (2 (105 cells/ml) of uninfected PM1 cells or PM1cells chronically infected with MV3-HXB2 virus. After 3 days,photomicrographs of cultures were taken and cell viability was measuredby adding of 50 (l of 1 mg/ml2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilidesolution containing 20 (M phenazine methosulfate and recording the OD at450 nm. Data are expressed as the percentage inhibition of cytopathicity[calculated as 100% ((R (V)/(U (V), where U, V, and R represent ODvalues obtained for HOS-CD4.CCR5 cells cultured with uninfected PM1cells, or with HIV-1-infected cells in the absence or presence ofchemokine, respectively].

EXAMPLE 2 RANTES, MCP-2, Eotaxin, and IP-10 Are Substrates of CD26

ES-MS analysis revealed that 100 nM rhRANTES underwent partial tocomplete hydrolysis when incubated overnight at 37° C. with increasingamounts (25 to 250 (U) of sCD26 (FIG. 1). Taking into accountcationization (K+) of the multiply charged ions, the measured molecularmasses of the native and degraded polypeptides corresponded to thetheoretical masses of full-length (residues 1 to 68) and truncated(residues 3 to 68) forms of RANTES, respectively. The calculateddifference between the molecular masses of the native and the truncatedforms ranged from 183 to 185 daltons, which is consistent with theexpected mass (184 daltons) of a released Ser-Pro dipeptide, thepredicted NH2-terminus of RANTES (16). In contrast to the effect ofenzymatically active sCD26, shortened RANTES was not generated byincubation of the chemokine with a mutant sCD26 deficient in enzymeactivity (FIG. 1). RANTES also inhibited, possibly in a competitivemanner, the rapid hydrolysis of a pNA-conjugated Gly-Pro dipeptide byhuman placental DPPIV, as measured in a colorimetric enzyme assay (FIG.2). The efficacy of inhibition by chemically synthesized RANTES(1-68)was similar to that observed with the DPPIV substrate and competitiveinhibitor Ile-Pro-Ile (Diprotin A) (17), whereas RANTES (3-68) did notinhibit the reaction.

EXAMPLE 3 Sensitivity to CD26-mediated Cleavage

Sensitivity to CD26-mediated cleavage was not a unique property ofRANTES (Table 1.). Cleavage products with the predicted molecular masseswere also evident in samples of MCP-2, eotaxin and IP-10 afterincubation with sCD26. In contrast, MCP-1, which has a 62% sequencesimilarity with MCP-2 including the NH2-terminal QP dipeptides, was notcleaved by the enzyme under the same experimental conditions.

EXAMPLE 4 CD26-specific Truncation of RANTES Modifies Its Target CellSpecificity

To investigate the functional significance of DPPIV-mediated truncationof RANTES, we compared the effects of chemically synthesizedRANTES(1-68) and RANTES(3-68) on monocytes and monocyte-derivedmacrophages. Both resting cells and cells activated with M-CSF wereanalyzed because RT-PCR revealed marked changes in the abundance ofchemokine receptor transcripts in response to M-CSF activation (FIG. 3).In resting cells, transcripts encoding the chemokine receptors CCR1,CCR2b, or CXCR4, as well as control glyceraldehyde phosphatedehydrogenase (GAPDH) mRNA, were readily detectable, whereas CCR5receptor transcripts were virtually absent. After differentiation tomacrophages, the intensity of the CXCR4 and GAPDH signals remainedvirtually unchanged, whereas the abundance of CCR1 and CCR5 mRNAsincreased substantially and the CCR2b transcript virtually disappeared.CCR3 mRNA was not detected in either cell type.

Transient changes in the cytosolic free Ca2+ concentration ([Ca2+]i)were recorded after stimulation of monocytes or macrophages with anoptimal concentration of RANTES(1-68) or RANTES(3-68), and the effectswere compared with those of other chemokines (FIG. 4). Addition of 100nM RANTES(1-68) to cells loaded with the fluorescent Ca2+ probe Fura-2induced a rapid increase in [Ca+]i in both monocytes and macrophages. Incontrast, the same concentration of RANTES(3-68) increased [Ca2+]i inmacrophages but not in monocytes. Among the other chemokines tested,macrophage inflammatory protein-1((MIP-1( ), monocyte chemotacticprotein-1( ) (MCP-1), MCP-3 (1, 6), and stromal-derived factor-1((SDF-10(18-20) also increased [Ca2+]i in resting monocytes, whereas MCP-2 (21)induced a barely detectable response and MIP-1((1, 6) was inactive. Onthe basis of the previously described receptor specificities of thesechemokines (1, 6, 19, 20), the obtained activity pattern is consistentwith expression of CCR1, CCR2b, and CXCR4 receptors on monocytes (FIG.3). Macrophages showed marked Ca2+ responses to MIP-1(, MIP-1(, MCP-2,MCP-3, and SDF-1(, but were resistant to MCP-1, consistent with thepresence of transcripts encoding CCR1, CCR5, and CXCR4, and the absenceof those encoding CCR2b, in these cells (FIG. 3).

EXAMPLE 5 RANTES(3-68) Is a Chemokine Agonist, with Altered ReceptorSpecificity

Agonists that act at common chemokine receptors block each other'sactivity as a result of receptor desensitization, whereas responses tochemokines that act at different receptors are generally not affected(1, 6). We therefore performed comparative desensitization experimentsto define the types of receptors that mediate the effects of nativeversus truncated RANTES in macrophages (FIG. 5). Macrophages that werestimulated first with 100 nM RANTES(1-68) did not exhibit a second Ca2+response when challenged with the same dose of either full-length ortruncated RANTES. In contrast, cells stimulated with 100 nM RANTES(3-68)fully retained their ability to respond to a subsequent challenge withfull-length RANTES, but were desensitized to the effect of the truncatedform. These results suggest that the receptor repertoire available fortruncated RANTES is more restricted than that available for the nativechemokine. To characterize further the receptor usage of the differentforms of RANTES and other chemokines, we also studied the sensitivity ofMIP-1(-, MCP-3-, and SDF-1(-induced Ca2+ responses to RANTES-mediatedreceptor desensitization (FIG. 5). Of the known receptors, RANTESsignals via CCR1, CCR4, and CCR5, whereas MIP-1(acts at CCR5 exclusivelyand MCP-3 binds only to CCR1 and CCR2b at the concentrations used in ourexperiments (1, 6). The only receptor known to bind SDF-1(is CXCR4 (19,20). Pretreatment of macrophages with full-length RANTES blocked theability of MIP-1(and MCP-3, but not that of SDF-1(, to increase [Ca2+]i.In contrast, RANTES(3-68) desensitized cells to the effect of MIP-1(butdid not affect the response to MCP-3 or SDF-1. These results areconsistent with previous data on RANTES-induced receptor desensitization(1) and with our data on chemokine receptor mRNA abundance (FIG. 3).They suggest that, in M-CSF-activated macrophages, full-length RANTESshares CCR1 and CCRS receptors with MCP-3 and MIP-1(, respectively. Ourresults also indicate that, without its two NH2-terminal residues,RANTES is still able to signal via CCR5 but can no longer act at theCCR1 receptor.

EXAMPLE 6 CCR1- and CCR5-mediated Signaling of RANTES

HEK-293 cells expressing CCR5 and HOS-CD4 cells expressing CCR1 wereloaded with Fura-2 and exposed to various concentrations of RANTES(1-68)or RANTES(3-68). The two RANTES variants showed similar abilities toincrease [Ca2+]i in the CCR5 transfectant (FIG. 6A); the responses weredose dependent, with 10 nM of each variant sufficient to induce amaximal Ca2+ response. In contrast, in the cells expressing CCR1, theamount of RANTES(3-68) required to produce a detectable Ca2+ responsewas ˜100 times that for RANTES (1-68) (FIG. 6B); the effect ofRANTES(1-68) saturated at 50 nM, whereas that of RANTES(3-68) appearednot to have achieved saturation at 200 nM. Furthermore, bidirectionalcross-desensitization between the two RANTES variants was evident onlywith the cells expressing CCR5 (FIG. 6C); in the CCR1 transfectant,cross-desensitization was induced by full-length RANTES but not by thetruncated form, which also did not exhibit self-desensitization (FIG.6D). Control cells transfected with vector alone or with vectorsencoding CCR2b, CCR3, or CXCR4 did not respond to these ligands (datanot shown). These results thus confirm that the native andCD26-truncated RANTES variants exhibit markedly different activities atthe CCR1 receptor.

EXAMPLE 7 RANTES(3-68) Is a Potent Inhibitor of HIV-1

In addition to their function in chemotaxis, RANTES, MIP-1(, andMIP-1(each inhibit HIV-1 infection by competitive binding to CCR5(22-27), and this inhibition does not require receptor-mediated cellsignaling (27, 28). To examine whether removal of the two NH2-terminalresidues affects the antiviral activity of RANTES, we mixed HOS-CD4cells expressing recombinant CCR5 and PM1 cells chronically infectedwith the M-tropic recombinant MV3-HXB2 virus and cocultured them in theabsence or presence of various concentrations of RANTES(1-68) or RANTES(3-68). Both RANTES variants inhibited HIV-1-induced syncytium formationand cytopathicity (FIG. 7). Thus, similar to signaling activity throughCCR5, competitive inhibition of HIV-1 infection does not require theNH2-terminal Ser-Pro residues of RANTES.

The CD26 cleavage product of RANTES, RANTES(3-68), acts as a chemokineagonist with altered receptor-specificity. Hydrolysis by CD26 mightexplain why RANTES(3-68) has been isolated as a second component inaddition to intact RANTES from culture supernatants of stimulated humanfibroblasts, skin samples, and platelet preparations (29, 30). TheCC-chemokines RANTES, MCP-2, and eotaxin, and the CXC-chemokine IP-10are the first immune modulators and the longest polypeptides identifiedas natural substrates for CD26.

CD26 exists in both soluble and membrane-expressed forms. Secreted formsof CD26 have been identified in cell cultures and in human serum (31,32), although CD26 may be more active when expressed as an ectoenzyme athigh concentrations on endothelial cells, hepatocytes, kidney brushborder membranes, and leukocytes (10). Up-regulation of CD26 expressionon T lymphocytes and macrophages has been linked to cell activation anddevelopment of immunological memory (10). Thus, activation-inducedchanges in CD26 expression could affect the course of an inflammatoryresponse by modifying the target cell specificity of RANTES or otherchemokines, and by regulating the equilibrium between the migrating cellsubsets. We are currently addressing whether cells with different levelsof CD26 expression (e.g. naive versus memory T cells) secrete truncatedforms of RANTES or other chemoattractants, or are capable of modifyingexogenous chemokines.

The differential effects of CD26-truncated RANTES on monocytes versusmacrophages illustrate a role for cell differentiation in regulatingchemokine sensitivity through altered receptor expression. Ourfunctional and receptor transcript data indicate that CCR1 and CCR2b maybe the two principal CC chemokine receptors in resting monocytes,although other unidentified and functionally overlapping receptors mayalso contribute to chemokine function. Cell differentiation markedlychanges the pattern of chemokine sensitivity by reducing CCR2bexpression, thereby rendering the cells resistant to MCP-1, whileincreasing CCR5 expression, thereby augmenting the responses toCD26-truncated RANTES and MIP-1(. An increase in CCR5 expression alsomay render macrophages more susceptible to infection by M-tropicvariants of HIV-1. We have shown that macrophages also express CXCR4,the coreceptor for T cell line-tropic HIV-1 variants (33-34), asassessed by receptor transcript abundance and functional activity of theCXCR4 ligand SDF-1(. Nevertheless, activated macrophages are relativelyresistant to infection by T cell line-tropic HIV-1 variants (35), whichsuggests that factors other than CXCR4 may also be required forefficient infection of macrophages by these types of viruses.

Removal of two NH2-terminal residues by CD26 abolishes the interactionof RANTES with CCR1, but does not affect the anti-HIV-1 activity or theCCR5 signaling properties of the chemokine. Proline residues alsoinfluence the susceptibility of proximal peptide bonds to proteolyticenzymes (6), and so the removal of such residues by CD26 may also reducethe half-life of RANTES and other chemokines during an inflammatoryresponse. It will be important to determine whether CD26-mediatedcleavage is a general mechanism for changing the receptor specificityand functional activity of other chemokines, including those examined inthis study (MCP-2, eotaxin, and IP-10).

Many, but not all CC- and CXC-chemokines contain X-Pro- orX-Ala-amino-terminal sequence and are potential substrates of DPPIV. Weare currently exploring whether the inability of CD26 to cleave MCP-1 isdue to aggregation of this chemokine under these experimental conditionsor to a conformational requirement of the enzyme that is not fulfilledby MCP-1. Selectivity of CD26 activity on chemokines may function toreduce redundancy in chemokine target cell specificity as illustrated bythe different activity of full-length and truncated RANTES on monocytesversus macrophages. Finally, truncated analogs of chemokines withselective activity on distinct functional receptors, or analogs thatresist CD26 cleavage, may prove therapeutically beneficial in blockingor inducing the infiltration of specific subsets of effector cellsmediating inflammation, allergy and anti-tumor responses.

TABLE 1 Chemokine cleavage products after digestion with sCD26.Molecular masses by mass spectrometry (Da) NH2-terminal CD26 Full lengthTruncated Chemokine dipeptide cleavage Theoretical Observed TheoreticalObserved Eotaxin GP Yes 8361 8361 8207 8207 IP-10 VP Yes 8633 8637/8751*8437 8440/8555* MCP-1 QP No 8681 8678 8456 ND∥ MCP-2 QP Yes 8910 89098685 8686/8703# *Tentatively identified as [M + trifluoroacetic acid(TFA)]+; molecular mass of TFA is 114 Da. #Tentatively identified as[M + H2O]+. ∥ND = not detected.

REFERENCES

1. Murphy, P. M. 1996. Chemokine receptors: structure, function and rolein microbial pathogenesis. Cytokine Growth Factor Rev. 7:47-64.

2. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N. Wells, W.Luini, N. Polentarutti, S. Sozzani, and A. Mantovani. 1997. Bacteriallipopolysaccharide rapidly inhibits expression of C-C chemokinereceptors in human monocytes. J. Exp. Med. 185:969-974

3. Weber, M., M. Uguccioni, M. Baggiolini, I. Clark-Lewis, and C. A.Dahinden. 1996. Deletion of the NH2-terminal residue converts monocytechemotactic protein 1 from an activator of basophil mediator release toan eosinophil chemoattractant. J. Exp. Med. 183:681-685.

4. Gong, J.-H., M. Uguccioni, B. Dewald, M. Baggiolini, and I.Clark-Lewis. 1996. RANTES and MCP-3 antagonists bind multiple chemokinereceptors. J. Biol. Chem. 271:10521-10527.

5. Arenzana-Seisdedos, F., J.-L. Virelizier, D. Rousset, I. Clark-Lewis,P. Loetscher, B. Moser, and M. Baggiolini. HIV blocked by chemokineantagonist. 1996. Nature. 383:400.

6. Murphy, P. M. 1994. The molecular biology of leukocytechemoattractant receptors. Annu. Rev. Immunol. 12:593-633.

7. Walter, R., W. H. Simmons, and T. Yoshimoto. 1980. Proline specificendo- and exopeptidases. Mol. Cell. Biochem. 30:111-127.

8. Fox, D. A., R. E. Hussey, K. A. Fitzgerald, 0. Acuto, C. Poole, L.Palley, J. F. Daley, S. F. Schlossman, and E. L. Reinherz. 1984. Ta1, anovel 105 kD human T cell activation antigen defined by a monoclonalantibody. J. Immunol. 133:1250-1256.

9. Hegen, M., G. Niedobitek, C. E. Klein, H. Stein, and B. Fleischer.1990. The T cell triggering molecule Tp103 is associated with dipeptidylaminopeptidase IV activity. J. Immunol., 144:2908-2914.

10. Fleischer, B. 1994. CD26: a surface protease involved in T-cellactivation. Immunol. Today. 15:180-184.

11. Oravecz, T., G. Roderiquez, J. Koffi, J. Wang, M. Ditto, D. C.Bou-Habib, P. Lusso, and M. A. Norcross. 1995. CD26 expressioncorrelates with entry, replication and cytopathicity of monocytotropicHIV-1 strains in a T-cell line. Nature Med. 1:919-926.

12. Samson, M., O. Labbe, C. Mollereau, G. Vassart, and M. Parmentier.1996. Molecular cloning and functional expression of a new humanCC-chemokine receptor gene. Biochemistry. 35:3362-3367.

13. Tanaka, T., D. Camerini, B. Seed, Y. Torimoto, N. H. Dang, J.Kameoka, H. N. Dahlberg, S. F. Schlossman, and C. Morimoto. 1992.Cloning and functional expression of the T cell activation antigen CD26.J Immunol. 149:481-486.

14. Davis, S. J., H. A. Ward, M. J. Puklavec, A. C. Willis, A. F.Williams, and A. N. Barclay. 1990. High level expression in Chinesehamster ovary cells of soluble forms of CD4 T lymphocyte glycoproteinincluding glycosylation variants. J. Biol. Chem. 265:10410-10418.

15. McCaughan, G. W, J. E. Wickson, P. F. Creswick, and M. D. Gorrell.1990. Identification of the bile canalicular cell surface molecule GP110as the ectopeptidase dipeptidyl peptidase IV: an analysis by tissuedistribution, purification and N-terminal amino acid sequence.Hepatology. 11:534-544.

16. Schall, T. J., J. Jongstra, B. J. Dyer, J. Jorgensen, C. Clayberger,M. M. Davis, and A. M. Krensky. 1988. A human T cell-specific moleculeis a member of a new gene family. J. Immunol. 141:1018-1025.

17. Rahfeld, J., M. Schierhorn, B. Hartrodt, K. Neubert, and J. Heins.1991. Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu)inhibitors or substrates of dipeptidyl peptidase IV? Biochim. Biophys.Acta. 1076:314-316.

18. Nagasawa, T., H. Kikutani, and T. Kishimoto. 1994. Molecular cloningand structure of a pre-B-cell growth-stimulating factor. Proc. Natl.Acad. Sci. U.S.A. 91:2305-2309.

19. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J.Sodroski, and T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 382:829-833.

20. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J.-L. Virelizier,F. Arenzana-Seisdedos, O. Schwartz, J.-M. Heard, I. Clark-Lewis, D. F.Legler, M. Loetscher, M. Baggiolini, and B. Moser. 1996. The CXCchemokine SDF-1 is the ligand for LESTR/fusin and prevents infection byT-cell-line-adapted HIV-1. Nature. 382:833-835.

21. Van Damme, J., P. Proost, J.-P. Lenaerts, and G. Opdenakker. 1992.Structural and functional identification of two human, tumor-derivedmonocyte chemotactic proteins (MCP-2 and MCP-3) belonging to thechemokine family. J. Exp. Med. 176:59-65.

22. Alkathib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy,P. M. Murphy, and E. A. Berger. 1996. CC CKR5: A RANTES, MIP-1(, MIP-1(,receptor as a fusion cofactor for macrophage-tropic HIV-1. Science.272:1955-1958.

23. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath,L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J.Sodroski. 1996. The (-chemokine receptors CCR3 and CCR5 facilitateinfection by primary HIV-1 isolates. Cell. 85:1135-1148.

24. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C.Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. Adual-tropic primary HIV-1 isolate that uses fusin and the (-chemokinereceptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell.85:1149-1158.

25. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P.Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C.Peiper, T. J. Schall, D. R. Littman, and N. R. Landau. 1996.Identification of a major co-receptor for primary isolates of HIV-1.Nature. 381:661-673.

26. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A.Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A.Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokinereceptor CC-CKR-5. Nature 381:667-673.

27. Oravecz, T., M. Pall, and M. A. Norcross. 1996. (-chemoldneinhibition of monocytotropic HIV-1 infection: Interference with apostbinding fusion step. J. Immunol. 157:1329-1332.

28. Farzan, M., H. Choe, K. A. Martin, Y. Sun, M. Sidelko, C. R. Mackay,N. P. Gerard, J. Sodroski, and C. Gerard. 1997. HIV-1 entry andmacrophage inflammatory protein-1beta-mediated signaling are independentfunctions of the chemokine receptor CCR5. J. Biol. Chem. 272:6854-6857.

29. Mallet, A. I., and I. Kay. 1995. Characterization of chemokineproinflammatory proteins by combined liquid chromatography-massspectrometry. Biochem. Soc. Trans. 23:911-913.

30. Noso, N. M., Sticherling, J. Bartels, A. I. Mallet, E. Christophers,and J.-M. Schr÷der. 1996. Identification of an N-terminally truncatedform of the chemokine RANTES and granulocyte-macrophagecolony-stimulating factor as major eosinophil attractants released bycytokine-stimulated dermal fibroblasts. J. Immunol. 156:1946-1953.

31. Tanaka, T., J. S. Duke-Cohan, J. Kameoka, A. Yaron, I. Lee, E. F.Schlossman, and C. Morimoto. 1994. Enhancement of antigen-induced T-cellproliferation by soluble CD26/dipeptidyl peptidase IV. Proc. Natl. Acad.Sci. U.S.A. 91:3082-3086.

32. Duke-Cohan, J. S., C. Morimoto, J. A. Rocker, and S. Schlossman.1996. Serum high molecular weight dipeptidyl peptidase IV (CD26) issimilar to a novel antigen DPPT-L released from activated T cells. J.Immunol. 156:1714-1721.

33. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1entry cofactor: Functional cDNA cloning of a seven-transmembrane, Gprotein-coupled receptor. Science. 272:872-876.

34. Berson, J. F., D. Long, B. J. Doranz, J. Rucker, F. R. Jirik, and R.W. Doms. 1996. A seven-transmembrane domain receptor involved in fusionand entry of T-cell-tropic human immunodeficiency virus type 1 strains.J. Virol. 70:6288-6295.

35. Cheng-Mayer, C., M. Quiroga, J. W. Tung, D. Dina, and J. A. Levy.1990. Viral determinants of human immunodeficiency virus type 1 T-cellor macrophage tropism, cytopathogenicity, and CD4 antigen modulation. J.Virol. 64:4390-4398.

36. Wang, J. M., D. W. McVicar, J. J. Oppenheim, and D. J. Kelvin. 1993.Identification of RANTES receptors on human monocytic cells: competitionfor binding and desenzitization by homologous chemotactic cytokines. J.Exp. Med. 177:699-705.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

2 1 198 DNA Homo sapiens CDS (1)..(198) 1 tat tcc tcg gac acc aca ccctgc tgc ttt gcc tac att gcc cgc cca 48 Tyr Ser Ser Asp Thr Thr Pro CysCys Phe Ala Tyr Ile Ala Arg Pro 1 5 10 15 ctg ccc cgt gcc cac atc aaggag tat ttc tac acc agt ggc aag tgc 96 Leu Pro Arg Ala His Ile Lys GluTyr Phe Tyr Thr Ser Gly Lys Cys 20 25 30 tcc aac cca gca gtc gtc ttt gtcacc cga aag aac cgc caa gtg tgt 144 Ser Asn Pro Ala Val Val Phe Val ThrArg Lys Asn Arg Gln Val Cys 35 40 45 gcc aac cca gag aag aaa tgg gtt cgggag tac atc aac tct ttg gag 192 Ala Asn Pro Glu Lys Lys Trp Val Arg GluTyr Ile Asn Ser Leu Glu 50 55 60 atg agc 198 Met Ser 65 2 66 PRT Homosapiens 2 Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala ArgPro 1 5 10 15 Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser GlyLys Cys 20 25 30 Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg GlnVal Cys 35 40 45 Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn SerLeu Glu 50 55 60 Met Ser 65

What is claimed is:
 1. A substantially pure polypeptide having an aminoacid sequence as set forth in SEQ ID NO:2 which is a truncated form ofRANTES (1-68), said polypeptide comprising the amino acid sequence asset forth in SEQ ID NO:2.
 2. An isolated polynucleotide which encodes anamino acid sequence as set forth in claim
 1. 3. An isolatedpolynucleotide selected from the group consisting of a) SEQ ID NO:1; b)SEQ ID NO:1, wherein T can also be U; c) nucleic sequences fullycomplementary to SEQ ID NO:1; d) fragments of a), b), or c) that are atleast 15 bases in length and that will hybridize to DNA which encodesSEQ ID NO:2.
 4. An expression vector containing in operable linkage thepolynucleotide as in claim
 2. 5. A host cell containing the vector ofclaim
 4. 6. The host cell of claim 5, wherein the cell is a eukaryoticcell.
 7. A substantially pure polypeptide consisting of the amino acidsequence as set forth in SEQ ID NO:2.